Guide to Antibody Libraries

To identify an antibody that effectively targets a specific antigen, scientists and drug developers must sift through billions of potential antibodies to find ones that best bind the antigen. While this may sound like a monumental task, antibody libraries, or collections of antibodies with diverse binding capabilities, have made the search easier for scientists. “Antibody libraries have major advantages when it comes to identifying antibodies during urgent times of need, such as during the global pandemic when speed is a necessity,” says Crystal Richardson, Ph.D., Business Partnership Manager of Gene Synthesis at Azenta Life Sciences.

In this article, we discuss the types of antibody libraries, how they are constructed, antibody display technology, and available vendor platforms for antibody discovery.

Types of antibody libraries

Antibody libraries have a variety of origins. They can come from donors that have been exposed to the antigen (immune libraries), from donors that have not been exposed to the antigen of interest (naive libraries), or they can be generated from synthetic sources (synthetic or semi-synthetic libraries). “The choice between naive, immune, or synthetic antibody libraries depends on project goals,” says Renny Feldman, Ph.D., Site Head and Director of Antibody Discovery at Charles River Laboratories. “Additionally, hybrid approaches may combine elements based on project needs, considering factors like diversity and specificity,” says Feldman.

Immune antibody libraries

Immune libraries are constructed using V genes from immunized donors or previously infected individuals. After immunization, B cells are isolated from the individual and the genes for the antibody’s variable regions of the heavy and light chains are amplified and cloned into a display vector.

“Because of their origin, immune libraries have undergone affinity maturation and therefore have the potential to deliver high-affinity antibodies with relatively small libraries.” says John Cardone, Ph.D., Marketing Manager for Custom Antibody Services at Bio-Rad. However, a downside to immune libraries is that they are antigen specific and can’t be used to target other antigens.

Naive antibody libraries

To overcome the antigen-specificity of immune antibody libraries, naive antibodies libraries can be used with other antigens. “Naive libraries are universally applicable tools that can be deployed against a wide range of targets, but can yield hits with medium affinities,” says Feldman. These libraries need to be large in order to generate high affinity antibodies. Naive antibody libraries are created in a similar way as immune libraries, but they are sourced from donors that have not previously encountered the antigen.

Synthetic and semi-synthetic antibody libraries

Unlike both naive and immune antibody libraries, synthetic and semi-synthetic antibodies are designed computationally and are generated from DNA synthesis rather than donor sources. Semi-synthetic libraries are created by mutating at least one complementary determining region using oligonucleotides-directed mutagenesis. In contrast, synthetic libraries are fully synthetic and are built by in vitro gene synthesis and assembly.

“Synthetic antibody libraries can be generated without need for immunizations, achieving diversity that can be far beyond the level identified by immunization alone,” says Richardson. “These libraries allow researchers to rapidly screen large numbers of antibody variants to effectively identify rare clones with ideal functional properties such as high affinity and specificity.”

In addition to expanding the diversity of antibodies, synthetic antibodies can also facilitate the screening process. For example, “adjusting codon usage can improve the expression in bacteria, which is an essential step in phage display,” says Cardone.

Antibody display options

Antibody libraries are used in a process called biopanning, which involves displaying the library to the antigen, screening for high-affinity binders, and repeated rounds of in vitro evolution to generate the best binders.

A variety of in vitro display technologies enable scientists to screen antibody libraries against their target by “displaying” the antibodies on the surface of phage or yeast, or on ribosomes, for example. Once the antibody library has been generated, they are cloned into the appropriate vector so that they can be displayed to the antigen. For example, antibody genes are cloned into plasmids to fuse the antibody gene with yeast surface protein genes or to the phage coat protein gene.

The choice of display technology depends on many factors. “Phage display libraries tend to be the largest and contain the most diversity, and there are robust, well-established methods for successfully carrying out antibody discovery via phage display,” says Feldman.

However, phages cannot be used to display full IgG antibodies and successful binders (ex: scFvs) from phage display may not translate therapeutically. “scFv to IgG conversion can yield nonreactive or less reactive antibodies, so phage display screening poses challenges for therapeutic development where IgG is typically preferred,” says Richardson. Yeast cells, on the other hand, are better suited to display full-length IgG antibodies.

“It is possible to combine the two technologies to get the best of both worlds, i.e., a comprehensive output of leads from phage display followed by sorting and selection of leads that recognize the desired target(s) through yeast display,” says Cardone.

Platforms for antibody discovery

Life science companies offer a variety of antibody library platforms and services to facilitate antibody discovery. Below, we describe a few of these platforms and services.

• Azenta Life Sciences: Azenta offers trimer-controlled and NNK combinatorial DNA libraries that are often used in synthetic and semi-synthetic antibody library generation. These libraries have flexible species and antibody formats and are available as linear fragments or as cloned pools with sequence verification, says Richardson.
• Bio-Rad: Bio-Rad’s Pioneer™ Antibody Discovery Platform uses SpyDisplay technology and is one of the largest fully human, phage display libraries available for the discovery and optimization of biotherapeutic leads, according to Cardone. Bio-Rad also offers a custom recombinant monoclonal antibody generation service, based on HuCAL^® PLATINUM Library.
• Charles River Laboratories: Charles River has several phage display libraries and antibody engineering services driven by AI (Tumbler™). Their phage display libraries include the SuperHuman library, which has a diversity of over 76 billion sequences, the Tungsten library for nanobody discovery, and their newest library, Cosmic.

Find more antibody library services here